Project 3 proposes the positional cloning of 2 separate autosomal recessive disorders, Duchenne-like Muscular dystrophy (DLMD) and the peripheral neuropathy Charcot-Marie-Tooth Disease (CMT4A). The Duchenne phenotype is the most common muscular dystrophy and similarly the CMT phenotype represents on of the most common and genetically heterogeneous inherited disorders in man. Through Project 1 and Dr. Vance's laboratories we have previously demonstrated linkage to chromosome 13q12 for DLMD z=8.9, 0.03, D13S115) and recently localized CMT4A to 8q21 (z=9.2, 0.00, D8S164). Inbred families from Tunisia have provided family material for both diseases. While both disorder are present in local populations, the recessive inheritance pattern makes investigation difficult. Therefore these highly consanguineous families present a unique and powerful opportunity to study two autosomal recessive disorders that contribute to common phenotypes. We propose to use selected YACs, band specific microdissection libraries and somatic cell hybrid libraries to develop highly polymorphic microsatellites that will closely flank these genes. Recently through haplotype analysis we have reduced the region flanking the DLMD gene to 5cM. The YAC megabase library of CORE B will be the primary source along with Pulse Field Gel Electrophoresis (PFGE) to construct a physical map spanning the disease locus. Coding regions will be identified through various techniques including direct selection, identification of CpG rich clones and exon amplification. Both muscle an peripheral nerve cDNA libraries are available and already in use in the Program laboratories. Candidate genes will be isolated and tested for mutations using PFGE, heteroduplex and SSCP. Once these defects have been identified, work can begin on the contribution of these defects to the DMD and CMT phenotypes as well to basic physiologic mechanisms.